An extracellular polysaccharide derived from Lactobacillus rhamnosus strain BD 4047, its preparation method, and its application.

Abstract

The application provides an exopolysaccharide from lactobacillus rhamnosus strain BD 4047 and a preparation method and application thereof, and belongs to the technical field of microbial polysaccharides. The exopolysaccharide from lactobacillus rhamnosus strain BD 4047 is isolated from supernatant obtained by fermentation of lactobacillus rhamnosus strain BD 4047 in skimmed milk, and contains three monosaccharides of rhamnose, glucose and galactose; the average molecular weight is (1.419-1.735) x 10 6 Daltons. The exopolysaccharide has the effects of improving phagocytic activity of macrophages, improving secretion and NO release amount of TNF-alpha in macrophages, and simultaneously activating NF-kappa B signal pathway in macrophages, so as to achieve the effect of regulating immunity. Moreover, the exopolysaccharide is safe and non-toxic, and provides a new means for preparation of immunoregulatory drugs and treatment of immune-related diseases.

Description

Technical Field

[0001] This invention belongs to the field of microbial polysaccharide technology, specifically relating to an extracellular polysaccharide derived from Lactobacillus rhamnosus strain BD 4047, its preparation method, and its application. Background Technology

[0002] Polysaccharides are considered to be a broad-spectrum, non-specific immune booster that can enhance the cellular and humoral immune functions of host cells, such as activating macrophages, T cells, B cells, and NK cells. They can also activate complement and induce the production of interferon. Their role is to activate the body's non-specific defense mechanisms, and they have good therapeutic effects in antiviral, antitumor, and anti-radiation applications.

[0003] Based on their origin, polysaccharides can be classified into animal polysaccharides, plant polysaccharides, and microbial polysaccharides. Among them, microbial polysaccharides, produced by microbial metabolism of carbohydrates, are less studied due to their low yield. Taking *Lactobacillus rhamnosus* as an example, currently, almost all extracellular polysaccharides from this strain are extracted from self-synthesized culture media, with very few reports from edible culture media. Therefore, structural characterization and functional determination of extracellular polysaccharides from *Lactobacillus rhamnosus* is an important scientific problem to be solved in this field, and also an important indicator for evaluating the application potential of this strain. Thus, the development of new microbial polysaccharides is of great significance for enriching the variety of immune stimulants.

[0004] Lactobacillus rhamnosus is a known beneficial bacterium. Patent publication CN115216422A discloses that Lactobacillus rhamnosus possesses high extracellular polysaccharide production, acid and choline resistance, and resistance to gastrointestinal digestive enzymes, exhibiting effects such as improving glucose tolerance, controlling liver indices, controlling blood lipids, and lowering insulin levels. Patent publication CN111154676A discloses that extracellular polysaccharides derived from Lactobacillus rhamnosus have strong antioxidant capacity, lipase inhibitory capacity, and lipid-lowering effects. Patent publication CN106635924A discloses that extracellular polysaccharides derived from Lactobacillus rhamnosus promote macrophage proliferation and have therapeutic effects on colitis. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide an extracellular polysaccharide derived from Lactobacillus rhamnosus strain BD 4047, which can enhance the phagocytic activity of macrophages and promote the secretion or release of TNF-α and NO, thereby achieving the purpose of immune regulation by activating the NF-κB signaling pathway.

[0006] This invention provides an extracellular polysaccharide derived from *Lactobacillus rhamnosus* strain BD 4047. The repeating units of the extracellular polysaccharide comprise the following monosaccharides: rhamnose, glucose, and galactose; the molar ratio of rhamnose, glucose, and galactose is (3.67–3.72):(1.47–1.52):1; and the average molecular weight is (1.419–1.735) × 10⁻⁶. 6 Dalton.

[0007] Preferably, the extracellular polysaccharide is composed of a main chain formed by alternating 1,3-linked rhamnose residues, 1,2,3-linked rhamnose residues, 1,2-linked glucose residues, and 1,3-linked glucose residues, with branched side chains formed at the O2 position of the 1,2,3-linked rhamnose residues. The branched side chains are formed by galactose residues with pyruvate substituents at their ends.

[0008] Preferably, the repeating unit of the extracellular polysaccharide is as shown in Formula I:

[0009]

[0010] This invention provides a method for preparing the extracellular polysaccharide, comprising the following steps;

[0011] The fermentation supernatant of Lactobacillus rhamnosus strain BD 4047 was precipitated with alcohol, and the precipitate was collected.

[0012] The precipitate was dissolved in water and mixed with trichloroacetic acid. The supernatant was collected to obtain crude polysaccharide.

[0013] The crude polysaccharide was separated by gel column chromatography, and the earliest appearing component peak was collected to obtain the extracellular polysaccharide.

[0014] A preferred method for preparing fermentation supernatant of Lactobacillus rhamnosus strain BD 4047 includes inoculating Lactobacillus rhamnosus strain BD 4047 into skim milk and allowing it to ferment statically, inactivating the fermented milk, separating the solid and liquid phases, and collecting the liquid phase as fermentation supernatant.

[0015] Preferably, the skim milk comprises skim milk powder and water;

[0016] The mass percentage of the skim milk powder to the mass percentage of the skim milk is 6% to 12%.

[0017] The inoculum size of *Lactobacillus rhamnosus* was 1.25 × 10⁻⁶. 7 ~1×10 8 CFU / mL;

[0018] The temperature for static fermentation is 25℃~45℃; the time for static fermentation is 12~36h.

[0019] Preferably, the volume ratio of the fermentation supernatant of the Lactobacillus rhamnosus strain BD 4047 to alcohol is 1:(3-4);

[0020] The final mass concentration of the trichloroacetic acid is 5% to 9%.

[0021] Preferably, during the gel column chromatography separation,

[0022] The gel column was filled with Sepharose 6FastFlow; the gel column had dimensions of 2.6cm × 30cm.

[0023] The eluent was a 0.2M NaCl aqueous solution;

[0024] The flow rate of the eluent is 0.2–0.3 mL / min.

[0025] This invention provides the application of the extracellular polysaccharide or the extracellular polysaccharide prepared by the preparation method in the preparation of immunomodulatory products.

[0026] Preferably, the immune modulation includes at least one of the following:

[0027] Enhance the phagocytic activity of macrophages;

[0028] Promotes the secretion of TNF-α by macrophages;

[0029] Promotes the release of NO from macrophages;

[0030] Activate the NF-κB signaling pathway.

[0031] This invention provides the application of the extracellular polysaccharide or the extracellular polysaccharide prepared by the preparation method in the preparation of macrophage function promoters.

[0032] This invention provides an extracellular polysaccharide derived from *Lactobacillus rhamnosus* strain BD 4047. Structural characterization of the extracellular polysaccharide produced by *Lactobacillus rhamnosus* fermentation of skim milk shows that the extracellular polysaccharide comprises the following monosaccharides: rhamnose, glucose, and galactose; the molar ratio of rhamnose, glucose, and galactose is (3.67–3.72):(1.47–1.52):1; and the average molecular weight is (1.419–1.735) × 10⁻⁶. 6 Dalton. The extracellular polysaccharide enhances macrophage phagocytic activity, increases TNF-α secretion and NO release in macrophages, and activates the NF-κB signaling pathway in macrophages, thereby achieving an immune-regulating effect. Furthermore, the extracellular polysaccharide is safe and non-toxic, providing a new approach for the preparation of immunomodulatory drugs and the treatment of immune-related diseases. Attached Figure Description

[0033] Figure 1Gel column chromatography pattern of extracellular polysaccharides derived from Lactobacillus rhamnosus strain BD 4047;

[0034] Figure 2 The 1H-NMR spectrum of extracellular polysaccharides derived from Lactobacillus rhamnosus strain BD 4047;

[0035] Figure 3 The 13C-NMR spectrum of extracellular polysaccharides derived from Lactobacillus rhamnosus strain BD 4047;

[0036] Figure 4 HSQCAD map of extracellular polysaccharides derived from Lactobacillus rhamnosus strain BD 4047;

[0037] Figure 5 gHMBCAD map of extracellular polysaccharides derived from Lactobacillus rhamnosus strain BD 4047;

[0038] Figure 6 NOESY map of extracellular polysaccharides derived from Lactobacillus rhamnosus strain BD 4047;

[0039] Figure 7 gCOSY map of extracellular polysaccharides derived from Lactobacillus rhamnosus strain BD 4047;

[0040] Figure 8 TOCSY map of extracellular polysaccharides derived from Lactobacillus rhamnosus strain BD 4047;

[0041] Figure 9 Cytotoxicity assay of extracellular polysaccharides derived from Lactobacillus rhamnosus strain BD 4047;

[0042] Figure 10 The effect of extracellular polysaccharides derived from Lactobacillus rhamnosus strain BD 4047 on phagocytic activity;

[0043] Figure 11 The effect of extracellular polysaccharides derived from Lactobacillus rhamnosus strain BD 4047 on NO release;

[0044] Figure 12 The effect of extracellular polysaccharides derived from Lactobacillus rhamnosus strain BD 4047 on TNF-α production;

[0045] Figure 13 The effect of extracellular polysaccharides derived from Lactobacillus rhamnosus strain BD 4047 on NF-κB activation.

[0046] Biological Preservation Information

[0047] This invention provides Lactobacillus rhamnoides strain BD 4047, which was deposited on November 15, 2016, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 13310. Detailed Implementation

[0048] This invention provides a method for preparing extracellular polysaccharides derived from Lactobacillus rhamnosus strain BD 4047, comprising the following steps;

[0049] The fermentation supernatant of Lactobacillus rhamnosus strain BD 4047 was precipitated with alcohol, and the precipitate was collected.

[0050] The precipitate was dissolved in water and mixed with trichloroacetic acid. The supernatant was collected to obtain crude polysaccharide.

[0051] The crude polysaccharide was separated by gel column chromatography, and the earliest appearing component peaks were collected and combined to obtain the extracellular polysaccharide.

[0052] In this invention, the fermentation supernatant of Lactobacillus rhamnosus strain BD 4047 is mixed with anhydrous ethanol, and the precipitate is collected.

[0053] In this invention, the Lactobacillus rhamnosus strain BD 4047 has the accession number CGMCCNo.13310 and is disclosed in the patent publication number CN 112322554A.

[0054] In this invention, the method for preparing the fermentation supernatant of Lactobacillus rhamnosus strain BD 4047 preferably includes inoculating Lactobacillus rhamnosus strain BD 4047 into skim milk and allowing it to ferment statically, inactivating the fermented milk, separating the solid and liquid phases, and collecting the liquid phase as the fermentation supernatant.

[0055] In this invention, the skim milk preferably comprises fresh skim milk or artificially formulated skim milk. The raw materials for the artificially formulated skim milk include skim milk powder and water. The skim milk powder accounts for 6% to 12% of the mass of the skim milk, more preferably 8%. The preparation method of the artificially formulated skim milk may include the following steps: mixing skim milk powder and water, sterilizing at 95–125°C for 5–20 minutes, and cooling to obtain the final product. The inoculum size of the *Lactobacillus rhamnosus* strain BD 4047 is preferably 1.25 × 10⁻⁶. 7 ~1×10 8 CFU / mL; more preferably 2.5 × 10⁻⁶. 7 ~7.5×10 7 CFU / mL, the optimal value is 5×10⁻⁶. 7CFU / mL. The static fermentation is preferably anaerobic fermentation. *Lactobacillus rhamnosus* is an anaerobic bacterium that can grow and reproduce rapidly under static culture conditions. The static fermentation temperature is preferably 25℃~45℃, more preferably 30℃~40℃, and most preferably 37℃. The static fermentation time is preferably 12~36h, more preferably 18~30h, and most preferably 24h. The fermentation milk inactivation method is preferably heat inactivation, and the heat inactivation temperature is preferably 90~100℃. The solid-liquid separation method is preferably centrifugation. The centrifugation speed is preferably 8,000~12,000g, more preferably 9,000~11,000g, and most preferably 10,000g. The centrifugation time is preferably 8~12min, more preferably 9~11min, and most preferably 10min.

[0056] In this invention, the volume ratio of the fermentation supernatant of the *Lactobacillus rhamnosus* strain BD 4047 to the alcohol is preferably 1:(3-4), more preferably 1:3. The alcohol is preferably anhydrous ethanol. Anhydrous ethanol facilitates the precipitation of polysaccharide components in the fermentation supernatant.

[0057] After obtaining the precipitate, the present invention dissolves the precipitate in water and mixes it with trichloroacetic acid, collects the supernatant, and obtains crude polysaccharide.

[0058] In this invention, the final mass concentration of trichloroacetic acid is preferably 5% to 9%, more preferably 7%. The trichloroacetic acid is beneficial for removing residual protein. After obtaining the supernatant, it is preferable to remove residual trichloroacetic acid from the supernatant. The method for removing trichloroacetic acid from the supernatant is preferably dialysis. The molecular weight cutoff of the dialysis bag is preferably 14,000 Daltons. The dialysis time is preferably 72 hours. The water change frequency during dialysis is preferably once every 8 hours.

[0059] After obtaining the crude polysaccharide, the present invention performs gel column chromatography to separate the crude polysaccharide and collects the earliest appearing component peak fraction to obtain the extracellular polysaccharide.

[0060] In this invention, during gel column chromatography, the preferred packing material is Sepharose 6FastFlow; the preferred column size is 2.6 cm × 30 cm. The preferred eluent is a 0.2 M NaCl aqueous solution. The preferred flow rate of the eluent is 0.2–0.3 mL / min. After elution, the fraction of the earliest appearing peak is collected, preferably at a frequency of 1 mL / tube. The combined extracellular polysaccharides are preferably dialyzed again to remove residual small molecules, and then freeze-dried to obtain the extracellular polysaccharides.

[0061] In this invention, inoculum size, culture temperature, and culture time are all factors affecting the yield of extracellular polysaccharides. Experiments show that lower inoculum size (0.1%), lower fermentation temperature (below 10°C), and shorter fermentation time (6 h) are all detrimental to the production of extracellular polysaccharides.

[0062] In this invention, the extracellular polysaccharides prepared above were analyzed for molecular weight, monosaccharide composition, and linkage relationships. The repeating units of the extracellular polysaccharides derived from *Lactobacillus rhamnosus* strain BD 4047 included the following monosaccharides: rhamnose, glucose, and galactose; the molar ratio of rhamnose, glucose, and galactose was (3.67–3.72):(1.47–1.52):1; and the average molecular weight was (1.419–1.735) × 10⁻⁶. 6 Dalton. The extracellular polysaccharide is preferably composed of a main chain consisting of alternating 1,3-linked rhamnose residues, 1,2,3-linked rhamnose residues, 1,2-linked glucose residues, and 1,3-linked glucose residues, with branched side chains formed at the O2 position of the 1,2,3-linked rhamnose residues, wherein the branched side chains are formed by galactose residues with pyruvate substituents at the ends.

[0063] In this invention, the repeating unit of the extracellular polysaccharide is preferably as shown in Formula I:

[0064]

[0065] In this invention, the extracellular polysaccharide was subjected to toxicity assay using the MTT assay. The results showed that the extracellular polysaccharide promoted macrophage proliferation in a dose-dependent manner at both low concentrations (6.25–25 μg / mL) and high concentrations (50–200 μg / mL). Therefore, the extracellular polysaccharide of Lactobacillus rhamnosus strain BD 4047 (CGMCC No. 13310) is safe and non-toxic within the range of 6.25–200 μg / mL.

[0066] This invention provides the application of the extracellular polysaccharide or the extracellular polysaccharide prepared by the preparation method in the preparation of immunomodulatory products.

[0067] In this invention, the immune regulation preferably includes at least one of the following:

[0068] Enhance the phagocytic activity of macrophages;

[0069] Promotes the secretion of TNF-α by macrophages;

[0070] Promotes the release of NO from macrophages;

[0071] Activate the NF-κB signaling pathway.

[0072] This invention provides the application of the extracellular polysaccharide or the extracellular polysaccharide prepared by the preparation method in the preparation of macrophage function promoters.

[0073] In this embodiment of the invention, the effect of the extracellular polysaccharide on the phagocytic activity of macrophages was detected using the neutral red assay. The results showed that treatment of phagocytic cells with extracellular polysaccharide A at concentrations of 25–200 μg / mL significantly enhanced the phagocytic rate in a dose-dependent manner, with the maximum phagocytic capacity reaching 110% of the positive control group at 200 μg / mL. This demonstrates that the extracellular polysaccharide derived from *Lactobacillus rhamnosus* strain BD4047 can stimulate macrophages and enhance their phagocytic capacity.

[0074] In another embodiment of the present invention, the amount of NO released by macrophages by the extracellular polysaccharide was determined by the Griess method. High concentration (200 μg / mL) of Lactobacillus rhamnosus strain BD 4047 (CGMCC No. 13310) extracellular polysaccharide significantly promoted the release of NO from macrophages.

[0075] In another embodiment of the present invention, the level of TNF-α in macrophage culture supernatant was measured using an ELISA kit. The results showed that a moderate concentration (50 μg / mL) of extracellular polysaccharide could significantly promote the secretion of TNF-α.

[0076] In another embodiment of the present invention, the effect of extracellular polysaccharides on NF-κB activation was studied. The results showed that the extracellular polysaccharides could improve the overall expression level of NF-κB by activating the NF-κB signaling pathway, thereby achieving the effect of regulating immunity.

[0077] The following detailed description, in conjunction with embodiments, illustrates an extracellular polysaccharide derived from Lactobacillus rhamnosus strain BD 4047, its preparation method, and its applications. However, these descriptions should not be construed as limiting the scope of protection of this invention.

[0078] Example 1

[0079] 1. Materials and Methods

[0080] (a) Preparation of seed culture (fermentation strain): Lyophilized Lactobacillus rhamnosus strain BD 4047 (CGMCC No. 13310) was dissolved in a small amount of sterile distilled water. A loopful was used to streak a single colony onto MRS solid medium (purchased from Merck Co., Germany). After anaerobic incubation at 37°C for 48 h, a single colony was picked up and placed into 10 ml of MRS liquid medium (purchased from Merck Co., Germany). The colony was evenly dispersed in the liquid medium using a vortex mixer and anaerobic incubated at 37°C for 48 h. The colony was then inoculated into MRS liquid medium at a 2% (v / v) inoculum and anaerobic incubated at 37°C for 24 h. The culture was then centrifuged at 15,000 rpm for 10 minutes, the supernatant was discarded, and the cells were washed twice with sterile distilled water. The cells were then resuspended in the original culture volume of sterile distilled water to obtain the seed culture for fermentation. The concentration of the seed culture was 2.5 x 10⁻⁶. 9 CFU / mL.

[0081] (b) Preparation of skim milk: Mix 10% by weight of skim milk powder with distilled water until fully dissolved, sterilize at 125°C for 5 min, and cool to room temperature to obtain skim milk of the required concentration.

[0082] 2. Preparation of extracellular polysaccharides from Lactobacillus rhamnosus

[0083] Lactobacillus rhamnosus strain BD 4047 (CGMCC No. 13310) was aseptically inoculated into skim milk (10% by mass) at an inoculation rate of 2% (v / v, the volume percentage of seed liquid to fermentation broth, the same below). The mixture was anaerobically cultured at 37℃ for 24 h to obtain fermented milk. The fermented milk was then inactivated by heating, centrifuged at 10,000g for 10 min, and the supernatant was collected. Three volumes of anhydrous ethanol were added for precipitation, followed by another centrifugation at 10,000g for 10 min. The precipitate was dissolved in water, and trichloroacetic acid was added to achieve a final concentration of 7%. The mixture was refrigerated and allowed to stand overnight, followed by another centrifugation at 10,000g for 10 min. The supernatant was then placed in a dialysis bag with a molecular weight cutoff of 14,000 Daltons and dialyzed for 72 h, with water changes every 8 h. The solution in the bag was freeze-dried to obtain crude polysaccharide A (yield of 340 mg / L).

[0084] 100 mg of crude polysaccharide A was dissolved in 5 mL of 0.2 M NaCl solution and loaded onto a Sepharose 6 Fast Flow column (D2.6 cm × 30 cm, purchased from GE, USA). Elution was performed isocratically with 0.2 M NaCl solution at a flow rate of 0.25 mL / min, collecting 1 mL from each collection tube. The polysaccharide content in each collection tube was determined using the sulfuric acid-phenol method. The elution products of peak F1 were combined. Figure 1 The solution was placed in a dialysis bag with a molecular weight cutoff of 14,000 Daltons and dialyzed for 72 hours, with the water changed every 8 hours. The solution in the bag was then freeze-dried to obtain extracellular polysaccharide A.

[0085] Example 2

[0086] 1. Materials and Methods

[0087] (a) Preparation of seeds (fermentation strains): Same as in Example 1.

[0088] (b) Preparation of skim milk: Mix 12% by mass of skim milk powder with distilled water until fully dissolved, sterilize at 95°C for 20 min, and cool to room temperature to obtain skim milk of the required concentration.

[0089] 2. Preparation of Lactobacillus rhamnosus inoculum

[0090] Lactobacillus rhamnosus strain BD 4047 (CGMCC No. 13310) was aseptically inoculated into skim milk (12% by mass) at an inoculum of 0.5% (v / v) and cultured anaerobically at 25°C for 36 h to obtain fermented milk. The fermented milk was inactivated by heating, centrifuged at 12,000 g for 8 min, and the supernatant was collected. Three volumes of anhydrous ethanol were added to precipitate the precipitate, and the mixture was centrifuged again at 12,000 g for 8 min. The precipitate was dissolved in water, and trichloroacetic acid was added to achieve a final concentration of 9%. The mixture was refrigerated and allowed to stand overnight, and then centrifuged again at 12,000 g for 8 min. The supernatant was collected and placed in a dialysis bag with a molecular weight cutoff of 14,000 Daltons. Dialysis was performed for 72 h, with water changes every 8 h. The solution in the bag was freeze-dried to obtain crude polysaccharide B (yield of 316 mg / L).

[0091] The crude polysaccharide B was purified by column chromatography according to Example 1 to obtain extracellular polysaccharide B.

[0092] Example 3

[0093] 1. Materials and Methods

[0094] (a) Preparation of seeds (fermentation strains): Same as in Example 1.

[0095] (b) Preparation of skim milk: Mix 6% by mass of skim milk powder with distilled water until fully dissolved, sterilize at 100°C for 15 min, and cool to room temperature to obtain skim milk of the required concentration.

[0096] 2. Preparation of Lactobacillus rhamnosus inoculum

[0097] Lactobacillus rhamnosus strain BD 4047 (CGMCC No. 13310) was aseptically inoculated into skim milk (6% by mass) at an inoculum of 4% (v / v) and cultured anaerobically at 45°C for 12 h to obtain fermented milk. The fermented milk was inactivated by heating, centrifuged at 8,000g for 12 min, and the supernatant was collected. Three volumes of anhydrous ethanol were added to precipitate the precipitate, and the mixture was centrifuged again at 8,000g for 12 min. The precipitate was dissolved in water, and trichloroacetic acid was added to achieve a final concentration of 5%. The mixture was refrigerated and allowed to stand overnight, and then centrifuged again at 8,000g for 12 min. The supernatant was collected and placed in a dialysis bag with a molecular weight cutoff of 14,000 Daltons. Dialysis was performed for 72 h, with water changed every 8 h. The solution in the bag was freeze-dried to obtain crude polysaccharide C (yield of 324 mg / L).

[0098] The crude polysaccharide C was purified by column chromatography according to Example 1 to obtain extracellular polysaccharide C.

[0099] Example 4

[0100] 1. Materials and Methods

[0101] (a) Preparation of seeds (fermentation strains): Same as in Example 1.

[0102] (b) Preparation of skim milk: Mix 8% by mass of skim milk powder with distilled water until fully dissolved, sterilize at 120°C for 10 min, and cool to room temperature to obtain skim milk of the required concentration.

[0103] 2. Preparation of Lactobacillus rhamnosus inoculum

[0104] Lactobacillus rhamnosus strain BD 4047 (CGMCC No. 13310) was aseptically inoculated into skim milk (8% by mass) at an inoculum of 3% (v / v) and cultured anaerobically at 30°C for 18 h to obtain fermented milk. The fermented milk was inactivated by heating, centrifuged at 11,000 g for 9 min, and the supernatant was collected. Three volumes of anhydrous ethanol were added to precipitate the precipitate, and the mixture was centrifuged again at 11,000 g for 9 min. The precipitate was dissolved in water, and trichloroacetic acid was added to achieve a final concentration of 5%. The mixture was refrigerated and allowed to stand overnight, and then centrifuged again at 11,000 g for 9 min. The supernatant was collected and placed in a dialysis bag with a molecular weight cutoff of 14,000 Daltons. Dialysis was performed for 72 h, with water changes every 8 h. The solution in the bag was freeze-dried to obtain crude polysaccharide D (yield of 309 mg / L).

[0105] The crude polysaccharide D was purified by column chromatography according to Example 1 to obtain extracellular polysaccharide D.

[0106] Example 5

[0107] 1. Materials and Methods

[0108] (a) Preparation of seeds (fermentation strains): Same as in Example 1.

[0109] (b) Preparation of skim milk: Mix 9% by mass of skim milk powder with distilled water until fully dissolved, sterilize at 115°C for 12 min, and cool to room temperature to obtain skim milk of the required concentration.

[0110] 2. Preparation of Lactobacillus rhamnosus inoculum

[0111] Lactobacillus rhamnosus strain BD 4047 (CGMCC No. 13310) was aseptically inoculated into skim milk (9% by mass) at an inoculum of 1% (v / v) and cultured anaerobically at 40℃ for 30 h to obtain fermented milk. The fermented milk was inactivated by heating, centrifuged at 9,000g for 11 min, and the supernatant was collected. Three volumes of anhydrous ethanol were added to precipitate the precipitate, and the mixture was centrifuged again at 9,000g for 11 min. The precipitate was dissolved in water, and trichloroacetic acid was added to achieve a final concentration of 5%. The mixture was refrigerated and allowed to stand overnight, and then centrifuged again at 9,000g for 11 min. The supernatant was collected and placed in a dialysis bag with a molecular weight cutoff of 14,000 Daltons. Dialysis was performed for 72 h, with water changed every 8 h. The solution in the bag was freeze-dried to obtain crude polysaccharide E (yield of 330 mg / L).

[0112] The crude polysaccharide E was purified by column chromatography according to Example 1 to obtain extracellular polysaccharide E.

[0113] Comparative Example 1

[0114] By adjusting the inoculum amount, culture temperature, and culture time in Example 1, the following group of crude polysaccharides of Lactobacillus rhamnosus prepared by different methods were obtained, and the yield of crude polysaccharides of each group is shown in Table 3.

[0115] Table 3. Yields of crude polysaccharides from *Lactobacillus rhamnosus* prepared by different methods.

[0116]

[0117] Example 6

[0118] Molecular weight distribution of extracellular polysaccharides from Lactobacillus rhamnosus

[0119] Different molecular weight dextrans were used as standards: STD-1 (Mw = 5,000), STD-2 (Mw = 12,000), STD-3 (Mw = 50,000), STD-4 (Mw = 270,000), and STD-5 (Mw = 670,000). The above series of standard polysaccharides and extracellular polysaccharides A, B, C, D, and E were dissolved in the mobile phase (0.1 mol / L NaNO3 solution) to obtain 1 mg / mL solutions. These solutions were filtered through a 0.45 μm filter and analyzed using an Agilent 1100 high-performance liquid chromatograph. A standard curve was plotted with the logarithm (LgMw) of the standard polysaccharide molecular weight on the x-axis and retention time (tR) on the y-axis, yielding a linear regression equation between the logarithm of molecular weight and retention time. The molecular weight of the samples was calculated based on the regression equation, and the results are shown in Table 2 below.

[0120] Table 2. Molecular weight determination of extracellular polysaccharides from Lactobacillus rhamnosus

[0121]

[0122] Conclusion: The average molecular weight of the extracellular polysaccharide from Lactobacillus rhamnosus strain BD 4047 (CGMCC No. 13310) is 1.419 × 10⁻⁶. 6 ~1.735×10 6 Dalton.

[0123] Example 7

[0124] Determination of the monosaccharide composition of extracellular polysaccharides in Lactobacillus rhamnosus

[0125] (1) Hydrolysis of polysaccharide samples

[0126] 2.0 mg of extracellular polysaccharides A, B, C, D, and E were placed in separate ampoules, and 3 mL of 2 mol / L trifluoroacetic acid (TFA) was added. The ampoules were sealed and hydrolyzed at 110 °C for 5 h. After cooling, the hydrolysate was evaporated to dryness under reduced pressure at 45 °C. Methanol was added and the evaporation was repeated several times to remove excess TFA, yielding the extracellular polysaccharide hydrolysate.

[0127] (2) Derivatization of hydrolyzed samples and mixed monosaccharide standards

[0128] The above-mentioned extracellular polysaccharide hydrolysis products were dissolved in 1 mL of water to obtain the sample solution to be derivatized. 1 mL of the aforementioned sample solution or a mixed standard solution of nine monosaccharides (0.5 mg / mL: rhamnose, fucose, glucuronic acid, galactose, glucose, mannose, galacturonic acid, arabinose, and xylose) was added, along with 1 mL of 0.6 mol / L NaOH solution and 1 mL of 0.5 mol / L PMP methanol solution. The mixture was thoroughly mixed to completely dissolve the solid product, and then reacted in a 70°C oven for 100 min. After cooling to room temperature, 0.3 mol / L HCl was added dropwise to adjust to neutrality. The mixture was extracted three times with chloroform, and the aqueous phase was collected. The aqueous phase was filtered through a 0.45 μm filter membrane and then used for HPLC analysis.

[0129] (3) Chromatographic conditions

[0130] An Agilent 1260 high-performance liquid chromatograph (purchased from Agilent Technologies, USA) equipped with a DAD detector and an Agilent Eclipse XDB-C18 column (purchased from Agilent Technologies, USA) was used. The column temperature was set at 30℃, the injection volume at 20 μL, the mobile phase was acetonitrile:0.1 mol / L phosphate buffer (pH 6.8) = 16:84 (V / V), and the detection wavelength was 250 nm.

[0131] (4) Data Analysis

[0132] The types of monosaccharides in the polysaccharide samples were determined by referring to the retention times of different monosaccharide standards (purchased from Sigma-Aldrich, USA). Then, the molar ratio of each monosaccharide in the polysaccharide sample was determined based on the peak area ratio of each monosaccharide component. The results are shown in Table 3.

[0133] Table 3. Determination of monosaccharide composition of extracellular polysaccharides from Lactobacillus rhamnosus

[0134]

[0135] Conclusion: The extracellular polysaccharide of Lactobacillus rhamnosus strain BD 4047 (CGMCC No. 13310) is a heteropolysaccharide composed of rhamnose, glucose and galactose in a molar ratio of 3.67–3.72:1.47–1.52:1.

[0136] Example 8

[0137] Determination of the extracellular polysaccharide linkage mode of Lactobacillus rhamnosus

[0138] (1) Methylation analysis

[0139] Glucuronic acid reduction: Glucuronic acid was reduced using the carbodiimide-sodium borohydride method (EDC-NaBH4). The reaction consisted of two stages. Stage 1: 50 mg of extracellular polysaccharide A was dissolved in 6 mL of ultrapure water and stirred until completely dissolved. 500 mg of EDC was added to the solution in two portions, 30 min apart, with the pH maintained between 4.5 and 4.8 using 0.1 mol / L HCl. The entire reaction process took 3 h. Stage 2: 8 mL of 2 mol / L NaBH4 was added dropwise to the above system over 40 min, maintaining the pH at approximately 7.0. After the addition was complete, the reaction continued for 1 h. The product was then dialyzed against running water in a dialysis bag (molecular weight cutoff of 3500 Da) for 24 h. The above steps were repeated four times, and the complete reduction of glutaronic acid was detected by PMP-HPLC.

[0140] Methylation: Take 20 mg of the completely reduced, dry polysaccharide sample and place it in a 10 mL reaction flask. Quickly add 3 mL of anhydrous dimethyl sulfoxide at room temperature, seal the flask, and magnetically stir for 30 min. Dissolve the sample using ultrasound assistance. Then, quickly add 50 mg of dry NaOH powder, seal the flask, and stir until most of the NaOH dissolves. Place the flask in an ice bath for 5 min. Slowly add 1 mL of iodomethane dropwise over 30 min, and continue the reaction at room temperature in the dark with stirring for another 30 min. Finally, add 1 mL of ultrapure water to terminate the reaction. Dialyze the product in a dialysis bag under running water for 24 h, then evaporate to dryness and repeat the above steps. After multiple methylation cycles, take a small sample for infrared spectroscopy detection. If the polysaccharide sample is within the range of 3400-3000 cm⁻¹... -1 The disappearance of the OH stretching vibration absorption peak indicates that the polysaccharide sample has been completely methylated; if the sample is not completely methylated, the reaction needs to be continued until the sample is completely methylated.

[0141] Hydrolysis and acetylation: 2 mg of fully methylated extracellular polysaccharide A sample was placed in an ampoule, 3 mL of 2 mol / L TFA was added, and the ampoule was sealed. Hydrolysis was carried out at 110 °C for 4 h under sealed conditions. Then, methanol was added and the mixture was rotary evaporated under reduced pressure several times to completely remove the TFA. After rotary evaporation, 3 mL of ultrapure water was added to dissolve the solid, followed by the addition of 50 mg of NaBH4. The mixture was magnetically stirred at room temperature for 3 h. After the reaction, acetic acid was added until the solution was weakly acidic (pH = 5). Methanol was added and the mixture was rotary evaporated to dryness. This process was repeated several times to completely remove boric acid. The resulting solid was dried in an oven at 100 °C for 10 min, 3 mL of acetic anhydride was added, and the mixture was reacted at 100 °C for 100 min. After the reaction, toluene (3 mL) was added, and excess acetic anhydride was removed by multiple co-evaporations. The product was dissolved in chloroform (5 mL), extracted three times with ultrapure water (5 mL × 3), the chloroform layer was recovered, anhydrous sodium sulfate powder was added to remove water, the mixture was allowed to stand for 30 min, evaporated to dryness under reduced pressure, dissolved in 0.5 mL of chloroform, filtered through a 0.22 μm organic filter membrane, and analyzed by GC-MS.

[0142] GC-MS conditions: Instrument model: Agilent 7820A / 5977GC-MS (purchased from Agilent Technologies, USA); Column model: HP-5 capillary column; Temperature program: initial temperature 120℃, hold for 2 min, then increase to 250℃ at a rate of 5℃ / min, hold for 10 min; Split injection mode with a split ratio of 3:1; Injection volume: 1 μL. The mass spectrometer ion source was an EI source with a voltage of 70 eV and a temperature of 180℃.

[0143] Data analysis: By comparing the EI-MS spectrum obtained by GC-MS with the standard PMAA spectrum and combining the results of monosaccharide composition, it can be determined that the linkage mode of each sugar residue of the reduced extracellular polysaccharide A is 1,3-linked rhamnose residue, 1,2,3-linked rhamnose residue, 1,2-linked glucose residue, 1,3-linked glucose residue, and 1,4,6-linked galactose residue.

[0144] (2) Nuclear magnetic resonance spectroscopy analysis

[0145] 20 mg of extracellular polysaccharide A was dissolved in 0.5 mL of D2O, transferred to a clean NMR tube, and analyzed by chromatography on a 600 MHz NMR spectrometer (Bruker, Switzerland) to obtain 1H-NMR ( ). Figure 2 ), 13C-NMR ( Figure 3 ), HSQCAD Figure 4 ), gHMBCAD Figure 5 NOESY Figure 6 ), gCOSY Figure 7 TOCSY Figure 8 After comprehensive analysis of the chromatogram, it was found that the extracellular polysaccharide backbone of Lactobacillus rhamnosus strain BD 4047 (CGMCC No. 13310) consists of 1,3-linked rhamnosus residues, 1,2,3-linked rhamnosus residues, 1,2-linked glucose residues, and 1,3-linked glucose residues. The branch point is located at the O2 position of the 1,2,3-linked rhamnosus residues. The side chain consists of galactose residues with pyruvate substituents at the end. Its repeating unit composition is shown in Formula I.

[0146]

[0147] Example 9

[0148] Toxicity test of Lactobacillus rhamnosus extracellular polysaccharide (MTT method)

[0149] RAW264.7 cells were fed at a concentration of 1×10⁻⁶. 4Cells were seeded in 96-well plates at different concentrations and incubated overnight at 37°C with 5% CO2 saturated humidity. After aspirating the culture medium, 100 μL of fresh DMEM cell culture medium containing different concentrations (0, 6.25, 12.5, 25, 50, 100, 200 μg / mL) of extracellular polysaccharide A or LPS (1 μg / mL) was added to each well and incubated at 37°C with 5% CO2 saturated humidity for 24 h. Then, the culture medium was aspirated, and 30 μL of LMT (5 mg / mL) was added to each well. The cells were incubated at 37°C with 5% CO2 saturated humidity for 4 h. After aspirating the liquid from the wells, 200 μL of DMSO was added to dissolve formazan crystals. The absorbance of each well at 490 nm was measured using a microplate counter (Multiskan FC, China). This value indirectly reflects the number of viable cells. The results are shown below. Figure 9 As shown.

[0150] Conclusion: Compared with the control group, low concentrations of extracellular polysaccharide A (6.25–25 μg / mL) had no significant effect on the cell viability of RAW264.7 macrophages, while high concentrations of extracellular polysaccharide A (50–200 μg / mL) promoted macrophage proliferation in a dose-dependent manner. Therefore, the extracellular polysaccharide of *Lactobacillus rhamnosus* strain BD 4047 (CGMCC No. 13310) is safe and non-toxic within the range of 6.25–200 μg / mL.

[0151] Example 10

[0152] Immunomodulatory effects of Lactobacillus rhamnosus extracellular polysaccharides

[0153] (1) Effect of Lactobacillus rhamnosus extracellular polysaccharide on phagocytic activity (neutral red method)

[0154] RAW264.7 cells were fed at a concentration of 1×10⁻⁶. 4 Cells were seeded in 96-well plates at different concentrations and incubated overnight at 37°C with 5% CO2 saturated humidity. After aspirating the culture medium, 100 μL of fresh DMEM cell culture medium containing different concentrations (6.25, 12.5, 25, 50, 100, 200 μg / mL) of extracellular polysaccharide A or LPS (1 μg / mL) was added to each well and incubated at 37°C with 5% CO2 saturated humidity for 24 h. Then, 100 μL of neutral red solution (8%) was added to each well and incubated at 37°C with 5% CO2 saturated humidity for 1 h. The liquid in the wells was aspirated, and the cells were washed three times with PBS. Lysis buffer (glacial acetic acid: ethanol = 1:1) was added to lyse the cells. The absorbance of each well at 540 nm was measured using a microplate counter. The results are shown below. Figure 10 As shown.

[0155] Conclusion: Treatment of RAW264.7 cells with extracellular polysaccharide A at concentrations of 6.25–12.5 μg / mL resulted in phagocytic rates that were essentially the same as the control group. However, with increasing concentrations of extracellular polysaccharide A (25–200 μg / mL), the phagocytic rate significantly increased in a dose-dependent manner, reaching a maximum phagocytic capacity of 110% of the positive control group at 200 μg / mL. These results indicate that, at the experimental concentrations, extracellular polysaccharides from *Lactobacillus rhamnosus* strain BD 4047 (CGMCC No. 13310) can stimulate RAW264.7 cells and enhance their phagocytic capacity.

[0156] (2) Effect of Lactobacillus rhamnosus extracellular polysaccharide on NO release (Griess method)

[0157] RAW264.7 cells were loaded at 5 × 10⁻⁶. 5 Cells were seeded in 96-well plates at different concentrations (6.25, 12.5, 25, 50, 100, 200 μg / mL) of extracellular polysaccharide A or LPS (1 μg / mL) and incubated overnight at 37°C with 5% CO2. After aspirating the culture medium, 100 μL of fresh DMEM cell culture medium containing different concentrations (6.25, 12.5, 25, 50, 100, 200 μg / mL) was added to the wells and incubated at 37°C with 5% CO2 for 24 h. The culture supernatant was collected, mixed with an equal volume of Griess reagent, and incubated at room temperature for 10 min. The absorbance of each well at 540 nm was measured using a microplate counter. A standard curve was plotted using NaNO2 as a standard to calculate NO secretion. The results are as follows: Figure 11 As shown.

[0158] Conclusion: When the extracellular polysaccharide concentration of Lactobacillus rhamnosus strain BD 4047 (CGMCC No. 13310) reaches 200 μg / mL, it can significantly promote the release of NO from macrophages.

[0159] (3) Effect of Lactobacillus rhamnosus extracellular polysaccharide on TNF-α production

[0160] RAW264.7 cells were loaded at 5 × 10⁻⁶. 5 Cells were seeded in 96-well plates at different concentrations (6.25, 12.5, 25, 50, 100, 200 μg / mL) of extracellular polysaccharide A or LPS (1 μg / mL) and incubated overnight at 37°C with 5% CO2. After aspirating the culture medium, 100 μL of fresh DMEM cell culture medium containing different concentrations (6.25, 12.5, 25, 50, 100, 200 μg / mL) of extracellular polysaccharide A or LPS (1 μg / mL) was added to the wells and incubated at 37°C with 5% CO2 for 24 h. The culture supernatant was collected, and the level of TNF-α in the supernatant was measured using an ELISA kit. The TNF-α concentration was calculated using a standard curve. The results are shown below. Figure 12 As shown.

[0161] Conclusion: When the extracellular polysaccharide concentration of Lactobacillus rhamnosus strain BD 4047 (CGMCC No.13310) reaches 50 μg / mL, it can significantly promote TNF-α expression.

[0162] (4) Effect of Lactobacillus rhamnosus extracellular polysaccharide on NF-κB activation

[0163] RAW264.7 cells were loaded at 5 × 10⁻⁶. 5 Cells were seeded in 96-well plates at a concentration of [specific concentration not specified] and cultured overnight at 37°C with 5% CO2 saturated humidity. After aspirating the culture medium, 100 μL of fresh DMEM cell culture medium containing extracellular polysaccharide A (200 μg / mL) or LPS (1 μg / mL) was added to the wells and incubated at 37°C with 5% CO2 saturated humidity for 4 h. Cells were then fixed with 4% paraformaldehyde for 20 min, and the reaction was stopped by adding 5% BSA and incubating at room temperature for 1 h. After adding NF-κB p65 antibody, the cells were stored overnight at 4°C. Cells were then removed and treated with CY3-labeled secondary antibody and DAPI, and incubated at room temperature for 5 min. Each step was followed by washing three times with washing buffer for 5 minutes each time. Finally, images were captured using a confocal microscope (A1R, Nikon, Japan), and fluorescence intensity was measured. The results are shown below. Figure 13 As shown.

[0164] Conclusion: Extracellular polysaccharides from Lactobacillus rhamnosus strain BD 4047 (CGMCC No. 13310) can regulate immunity by activating the NF-κB signaling pathway and increasing the overall expression level of NF-κB.

[0165] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A type of Lactobacillus rhamnosus ( Lactobacillus rhamnosus The extracellular polysaccharide derived from strain BD 4047 is characterized by: The repeating units of the extracellular polysaccharide include the following types of monosaccharides: rhamnose, glucose, and galactose; The molar ratio of rhamnose, glucose, and galactose is 3.7:1.5:1; The average molecular weight is 1.577 × 10⁻⁶. 6 Dalton; The method for preparing the extracellular polysaccharide includes the following steps; A method for preparing fermentation supernatant of Lactobacillus rhamnosus strain BD 4047 includes inoculating the seed culture of Lactobacillus rhamnosus strain BD4047 into skim milk and allowing it to ferment statically. The resulting fermented milk is then inactivated, solid-liquid separation is performed, and the collected liquid phase is the fermentation supernatant. The skim milk comprises skim milk powder and water; the skim milk powder accounts for 10% of the total mass of the skim milk; the inoculum amount of *Lactobacillus rhamnosus* strain BD 4047 is 2% of the volume of the fermented milk, and the bacterial concentration of the *Lactobacillus rhamnosus* strain BD 4047 seed culture is 2.5 × 10⁻⁶. 9 CFU / mL; The temperature for static fermentation is 37°C; the time for static fermentation is 24 hours. The fermentation supernatant of Lactobacillus rhamnosus strain BD 4047 was precipitated with alcohol, and the precipitate was collected; the volume ratio of the fermentation supernatant of Lactobacillus rhamnosus strain BD 4047 to anhydrous ethanol was 1:

3. The precipitate was dissolved in water and then mixed with trichloroacetic acid. The supernatant was collected to obtain crude polysaccharide; the final mass concentration of the trichloroacetic acid was 7%. The crude polysaccharide was separated by gel column chromatography, and the earliest peak fraction was collected to obtain the extracellular polysaccharide. The gel column packing material was Sepharose 6 Fast Flow. The gel column dimensions were 2.6 cm × 30 cm. The eluent was 0.2 M NaCl aqueous solution. The flow rate of the eluent was 0.2~0.3 mL / min.

2. The extracellular polysaccharide according to claim 1, characterized in that, The repeating unit of the extracellular polysaccharide is formed by alternating main chains of 1,3-linked rhamnose residues, 1,2,3-linked rhamnose residues, 1,2-linked glucose residues, and 1,3-linked glucose residues, with branched side chains formed at the O2 position of the 1,2,3-linked rhamnose residues. The branched side chains are formed by galactose residues with pyruvate substituents at the ends.

3. The extracellular polysaccharide according to claim 2, characterized in that, The repeating unit of the extracellular polysaccharide is shown in Formula I: Equation I.

4. The method for preparing the extracellular polysaccharide according to any one of claims 1 to 3, characterized in that, Includes the following steps; A method for preparing fermentation supernatant of Lactobacillus rhamnosus strain BD 4047 includes inoculating Lactobacillus rhamnosus strain BD4047 into skim milk and allowing it to ferment statically, inactivating the fermented milk, separating the solid and liquid phases, and collecting the liquid phase as fermentation supernatant. The skim milk comprises skim milk powder and water; the skim milk powder accounts for 10% of the total mass of the skim milk; the inoculum size of *Lactobacillus rhamnosus* is 0.5 × 10⁻⁶. 8 CFU / mL; The temperature for static fermentation is 37°C; the time for static fermentation is 24 hours. The fermentation supernatant of Lactobacillus rhamnosus strain BD 4047 was precipitated with alcohol, and the precipitate was collected; the volume ratio of the fermentation supernatant of Lactobacillus rhamnosus strain BD 4047 to anhydrous ethanol was 1:

3. The precipitate was dissolved in water and then mixed with trichloroacetic acid. The supernatant was collected to obtain crude polysaccharide; the final mass concentration of the trichloroacetic acid was 7%. The crude polysaccharide was separated by gel column chromatography, and the earliest peak fraction was collected to obtain the extracellular polysaccharide. The gel column packing material was Sepharose 6 Fast Flow. The gel column dimensions were 2.6 cm × 30 cm. The eluent was 0.2 M NaCl aqueous solution. The flow rate of the eluent was 0.2~0.3 mL / min.

5. The use of the extracellular polysaccharide according to any one of claims 1 to 3 or the extracellular polysaccharide prepared by the preparation method according to claim 4 in the preparation of immunomodulatory products.

6. The application according to claim 5, characterized in that, The immune modulation includes at least one of the following: Enhance the phagocytic activity of macrophages; Promotes the secretion of TNF-α by macrophages; Promotes the release of NO from macrophages; Activate the NF-κB signaling pathway.

7. The use of the extracellular polysaccharide according to any one of claims 1 to 3 or the extracellular polysaccharide prepared by the preparation method according to claim 4 in the preparation of macrophage function promoters.

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